title: VEGF rescues cigarette smoking-induced human RPE cell death by increasing autophagic flux: implications of the role of autophagy in advanced age-related macular degeneration.
abstract: OBJECTIVE
  Cigarette smoking (CS) is the most consistent risk factor for advanced age-related macular degeneration (AMD). To verify the molecular basis for CS-induced RPE alterations, RPE cell survival levels after being exposed to CS in relation with VEGF expression and autophagic flux were evaluated.
  
  
  METHODS
  Cigarette smoking extract (CSE) was added to ARPE-19 cells and hydrogen peroxide (HP) was used as a pure oxidant control. Cell survival was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Cell survival analysis was performed after pretreatment with anti-VEGF or recombinant VEGF. The expression of VEGF-A, VEGF-R1/R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR. LC3B-I (microtubule-associated protein-1 inhibitors), LC3B-II, and phosphorylation of Akt or Erk were measured with Western blot. Autophagic flux was determined by increasing LC3B-II levels with inhibitors of lysosomal proteases.
  
  
  RESULTS
  Incubation with 5% CSE for 16 hours induced approximately 30% cell death, which was similar to cell death levels when exposed to concentrations of 200 μM HP. Pretreatment with anti-VEGF did not decrease cell survival under CSE, unlike the decrease in cell survival shown with HP. However, supplementation with VEGF rescued CSE-induced RPE cell death. Interestingly, CSE caused an increase in autophagic flux, which was augmented with VEGF pretreatment. Cigarette smoking extract also degraded the total amounts of Akt levels, and VEGF blunted CSE-induced phosphorylation of Erk.
  
  
  CONCLUSIONS
  Cigarette smoking extract, similar to HP, affects cell viability and induces expression of VEGF and its receptors. Increased autophagic flux accelerated by treatment of exogenous VEGF may have a role in rescuing CSE-induced RPE cell death.
authors.affiliation: Siloam Eye Hospital, Seoul, Korea.
authors.forename: Young Kwang
authors.initials: YK
authors.lastname: Chu
authors.forename: Sung Chul
authors.initials: SC
authors.lastname: Lee
authors.forename: Suk Ho
authors.initials: SH
authors.lastname: Byeon
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008268:Macular Degeneration / Q000503:physiopathology*; D010936:Plant Extracts / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000473:pathology; D015398:Signal Transduction / Q000187:drug effects; D012907:Smoking / Q000009:adverse effects*; D014026:Nicotiana / Q000737:chemistry; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D010936:Plant Extracts; D042461:Vascular Endothelial Growth Factor A; D006861:Hydrogen Peroxide; D051057:Proto-Oncogene Proteins c-akt
keywords: AMD; RPE cells; VEGF; autophagy; cigarette smoke